PROJECT SUMMARY Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous drug-resistant pathogen. The death toll due to S. aureus is the second highest amongst patients infected with nosocomial drug-resistant bacteria. In the absence of a vaccine, antibiotics are the most effective solution to pharmacologically combat bacterial infections. However, the discovery of novel drugs capable of bypassing microbial drug resistance is a formidable and complex challenge. Amongst promising concepts for drug discovery and development is the use of copper. Free copper ions lack therapeutic properties but, if coordinated to small molecules, can bestow antimicrobial activity to the ensuing complex. These complexes can mediate target directed attacks. Recently, copper/ligand complexes with potentially exploitable antibacterial properties have emerged from our and other laboratories, but the concept has not yet been systematically translated into high-throughput drug discovery screening. According to our exploratory proof-of-principle screen on S. aureus, compounds with copper-dependent activities are not rare, but the insufficient copper content of commonly used screening media prohibited their discovery during high-throughput screening (HTS) in the past. Therefore, we propose to incorporate a copper-activation component in drug discovery screens so that a greater repertoire of bioactivities within existing libraries becomes available. Our preliminary data suggest that novel and previously undetectable antimicrobials are readily discovered through application of this concept. In collaboration with Southern Research, a world renowned not- for-profit center for HTS and drug development, we propose to establish a discovery and development pipeline for copper-related bacterial inhibitors. For that purpose, we will conduct a pilot screen to demonstrate the utility and specificity of our novel primary screening assay (AIM1) and the quality of novel probes for potential improvement and advancement towards medicinal applications (AIM2). Finally, we propose to perform mode of action studies, investigate biological responses and detail mechanisms of metal-ligand interactions (AIM3) at both the cellular and the molecular level.